The Flow Cytometry Core at NEI provides flow cytometry analytical and sorting equipment and services to the NEI Intramural community. It utilizes and develops state-of-the-art sample preparation, data acquisition and analysis, and sorting procedures in collaborative research projects. Provides training to students, fellows, and principal investigators on sample preparation, staining, and post-sort handling. Assesses technical research needs and recommends recruitment of the appropriate staff and acquisition of the equipment needed to meet those needs. This year the National Eye Institute made a great investment in biosafety with the addition of a new sorter. A new BD FACSAria Fusion flow cytometer with a fully integrated biosafety cabinet was added to the core. This sorter meets the new NIH's operator and sample protection requirements as well as global standards for bioprotection. OPERATION OF THE CORE: This year, sixty-three individuals from thirteen different laboratories used the facility. These services and collaborative services were performed for 8 Principal Investigators (PIs) from 8 NEI labs (ERPD, LI, LRCMB, MSF, N-NRL, RN, OGVFB and UNGIRD), plus 3 PIs from 3 other institutes at NIH (NICHD, NIDCD and NIDDK). Over 17,700 samples were analyzed. More than twenty users received training how to operate the MACSQuant instrumentation. This year the core performed about 1,400 hours of sorting. Among the techniques now in use within the core are methods for phenotyping live cells, detecting gene expression, monitoring membrane and DNA content changes due to apoptosis or proliferation, measurement of intracellular proteins and quantification of soluble proteins. The work involving human tissues includes the sorting of peripheral blood mononuclear cells to study their cytokine production, genotype and DNA or RNA expression. The sources are blood, buffy coat, and white cells. Some analytical work had been done with eye fluids, eye tissue specimen, protein, and tears. No tissues were stored by the core. TRAINING: The availability of the Technical IRTA position continues to have a very positive effect on the Core operation. There is a great need for well-trained flow cytometry operators. Through the TechIRTA, the hours of operation of the facility have been expanded from 40 hr/week to 60 hr/week. The expanded hours of operation helped to reduce the backlog on the sorter schedule. The TechIRTA position has greatly enhanced the ability of the Core to use the cell sorter for high throughput multicolor analysis. This year the core hosted a summer student from the Diversity In Vision Research and Ophthalmology (DIVRO) summer internship program. The student worked in detection of IRBP specific T cells and plasma cells in retina and lymphoid tissue of mice with spontaneous uveitis. Several formal training sessions were offered by the core to the NEI community. These courses included: Introduction to Flow Cytometry, Advanced Techniques (Ex. Apoptosis applications), Analytical Instrument Operation and Data Analysis with FloJo Software. The Core Manager completed 25 hours of continuing education in management of core laboratories and 8 hours of training in management of Core facilities. The technical IRTA completed 40 hours of formal training outside the Institute. The Core does not obligate users to acknowledge the Core contribution in their publications. The following are examples of the publications that acknowledged the use of NEI Flow Cytometry Core resources: Joe MK1, Kwon HS, Cojocaru R, Tomarev SI. Myocilin regulates cell proliferation and survival. J Biol Chem. 2014 Apr 4;289(14):10155-67. doi: 10.1074/jbc.M113.547091. Epub 2014 Feb 22. Kim SY, Yang HJ, Chang YS, Kim JW, Brooks M, Chew EY, Wong WT, Fariss R, Rachel R, Cogliati T8, Qian H, Swaroop A. Deletion of aryl hydrocarbon receptor AHR in mice leads to subretinal accumulation of microglia and RPE atrophy. Invest Ophthalmol Vis Sci. 2014 Aug 26. pii: IOVS-14-15091. doi: 10.1167/iovs.14-15091. Satpute-Krishnan P, Ajinkya M, Bhat S, Itakura E, Hegde RS, Lippincott-Schwartz J. ER Stress-Induced Clearance of Misfolded GPI-Anchored Proteins via the Secretory Pathway. Cell. 2014 Jul 31;158(3):522-33. doi: 10.1016/j.cell.2014.06.026. Shobi Veleri, Souparnika H. Manjunath, Robert N. Fariss, Helen May-Simera, Matthew Brooks, Trevor A. Foskett, Chun Gao, Teresa A. Longo, Pinghu Liu, Kunio Nagashima, Rivka A. Rachel, Tiansen Li, Lijin Dong & Anand Swaroop. Ciliopathy-associated gene Cc2d2a promotes assembly of subdistal appendages on the mother centriole during cilia biogenesis. Nat Commun. 2014 Jun 20;5:4207. doi: 10.1038/ncomms5207. Johnson TV, DeKorver NW, Levasseur VA, Osborne A, Tassoni A, Lorber B, Heller JP, Villasmil R, Bull ND, Martin KR, Tomarev SI. Identification of retinal ganglion cell neuroprotection conferred by platelet-derived growth factor through analysis of the mesenchymal stem cell secretome. Brain. 2014 Feb;137(Pt 2):503-19. doi: 10.1093/brain/awt292. Epub 2013 Oct 30. Chong WP, Horai R, Mattapallil MJ, Silver PB, Chen J, Zhou R, Sergeev Y, Villasmil R, Chan CC, Caspi RR. IL-27p28 inhibits central nervous system autoimmunity by concurrently antagonizing Th1 and Th17 responses. J Autoimmun. 2014 May;50:12-22. doi: 10.1016/j.jaut.2013.08.003. Epub 2013 Sep 7. Horai R, Silver PB, Chen J, Agarwal RK, Chong WP, Jittayasothorn Y, Mattapallil MJ, Nguyen S, Natarajan K, Villasmil R, Wang P, Karabekian Z, Lytton SD, Chan CC, Caspi RR. Breakdown of immune privilege and spontaneous autoimmunity in mice expressing a transgenic T cell receptor specific for a retinal autoantigen. J Autoimmun. 2013 Aug;44:21-33. doi: 10.1016/j.jaut.2013.06.003. Epub 2013 Jun 28. Ma W, Cojocaru R, Gotoh N, Gieser L, Villasmil R, Cogliati T, Swaroop A, Wong WT. Gene expression changes in aging retinal microglia: relationship to microglial support functions and regulation of activation. Neurobiol Aging. 2013 Oct;34(10):2310-21. doi: 10.1016/j.neurobiolaging.2013.03.022. Epub 2013 Apr 19.